Therapeutic polyamine-amides

ABSTRACT

A compound of formula I optionally in the form of an acid addition salt ##STR1## in which formula: A represents a substituent which is: hydroxyl, O-alkyl, O-cycloalkyl, O-alkenyl, O-aryl, O-aralkyl, O-carbamate, O-carbonate, O-acyl or halogen 
     a is 0-5; 
     the substituents A are identical or different when a is more than 1; 
     B represents a C 1  -C 6  aliphatic hydrocarbon group optionally carrying one or more of the substituents: hydroxyl, amino, halogen, C 1  -C 4  alkoxy, C 1  -C 4  alkyl, aryloxy or carboalkoxy and optionally comprising one or more sites of unsaturation and/or one or more carbonyl groups or ketal derivatives thereof; 
     b is 0 or 1; 
     c, d, f and h, which may be identical or different, are 2, 3, 4, 5 or 6; and 
     i is 0 or 1 
     D, E and F, which may be identical or different, represent hydrogen or C 1  -C 4  alkyl or cycloalkyl, and 
     G and J which may be identical or different represent hydrogen, alkyl or cycloalkyl or G and J together with the nitrogen atom to which they are attached represent a saturated heterocyclic ring system.

This invention relates to polyamine amides of interest for the treatmentof cerebral disorders and in particular psychoses, senile dementia,ischaemia, stroke, hypoxia, aneurysm, epilepsy, Parkinson's disease,Alzheimer's disease, Huntington's chorea, and related syndromes andneurological disorders.

Accordingly, the present invention comprises a compound of formula Ioptionally in the form of an acid addition salt ##STR2## in whichformula: A represents a substituent which is: hydroxyl, O-alkyl,O-cycloalkyl, O-alkenyl, O-aryl, O-aralkyl, O-carbamate, O-carbonate,O-acyl or halogen

a is 0-5;

the substituents A are identical or different when a is more than 1;

B represents a C₁ -C₆ aliphatic hydrocarbon group optionally carryingone or more of the substituents: hydroxyl, amino, halogen, C₁ -C₄alkoxy, C₁ -C₄ alkyl, aryloxy or carboalkoxy and optionally comprisingone or more sites of unsaturation and/or one or more carbonyl groups orketal derivatives thereof;

b is 0 or 1;

c, d, f and h, which may be identical or different, are 2, 3, 4, 5 or 6;and

i is 0 or 1

D, E and F, which may be identical or different, represent hydrogen orC₁ -C₄ alkyl or cycloalkyl, and

G and J which may be identical or different represent hydrogen, alkyl orcycloalkyl or G and J together with the nitrogen atom to which they areattached represent a saturated heterocyclic ring system.

Typically: when A represents O-alkyl, the alkyl group is C₁ -C₄ alkyl;when A represents O-cycloalkyl the group is cyclohexyl; when Arepresents O-alkenyl the alkenyl group is a C₁ -C₄ alkenyl group such asethenyl, propenyl or butenyl; when A represents O-aryl, the aryl groupis a phenyl group; when A represents O-aralkyl, the aralkyl group is aC₇ -C₁₀ phenalkyl group; when A represents O-carbamate, the O-carbamategroup is of formula --O--CO--NR^(I) R^(II) wherein R¹ and R¹¹, which maybe identical or different, represent C₁ -C₄ alkyl groups; when Arepresents O-carbonate, the O-carbonate is of formula --O--CO--OR^(III)wherein R^(III) represents a C₁ -C₄ alkyl group; when A representsO-acyl, the O-acyl group is of formula --O--CO--R^(IV) wherein R^(IV)represents a C₁ -C₄ alkyl group e.g. methyl and when A representshalogen, the halogen is typically Cl, Br or F.

It will be appreciated that the compounds in which A represents asubstituent other than hydroxyl or halogen may act through conversion invivo to the corresponding compound in which the group or groups A arehydroxy groups. Indeed, the present invention extends in general tocompounds of formula (I) as defined above in which A is hydroxyl when inpro-drug form.

Although a may be 0-5, it is typically at least 1 and normally no morethan 3, mono or di substitution of the aromatic ring being preferred anddisposition of a mono substituent e.g. a hydroxyl group, at the 2, 3 or4 position in the ring being especially so.

The aliphatic hydrocarbon group B is typically unbranched and generallycontains no more than two carbon atoms intermediate between the aromaticring and the carbonyl group of the CONH(CH₂)_(c) -- function. It will beappreciated that, when present, substituents may be identical ordifferent. Sites of unsaturation may be present as double bonds(typically configurationally trans) or triple bonds or both. Groups ofthe following formula are of particular interest, R₁ R₂ and R₃representing hydrogen or one or more of the substituents hereinbeforedescribed as carried on the C₁ -C₆ aliphatic hydrocarbon group: --CHR₁--, --CHR₁ CHR₂ --, --CR₁ ═CR₂ --, --CHR₁ CHR₂ CHR₃ --, --CR₁ ═CR₂ CHR₃-- and --CHR₁ --CR₂ ═CR₃ --.

Although R₁, R₂ and R₃ may represent one or more of the hereinbeforeidentified substituents it is most usual for all to be hydrogen.

Usually no more than one carbonyl group or ketal derivative thereof ispresent in the group B (i.e. one carbon atom of the C₁₋₆ aliphatichydrocarbon group is substituted by an oxo group or such a group inketal form) and there is generally at least one other carbon atompresent in the carbon chain between the aromatic ring and theCONH(CH₂)_(c) -- function as in the phenylpyruvic andhydroxyphenylpyruvic moieties wherein B represents --CH₂ CO--, which areof particular interest. When a carbonyl group is derivatised as a ketalgroup, of formula --C(OR_(a))(OR_(b))--, R_(a) and R_(b), which may beidentical or different, represent C₁ -C₆ alkyl groups e.g. methyl orethyl groups or groups which are linked together to form a ringincorporating the carbon and two oxygen atoms as for example in theethylene ketal wherein R_(a) and R_(b) each represent --CH₂ --.

D, E and F may be C₁₋₄ alkyl or, for example, C₃ -C₆ cycloalkyl but itis generally preferred for D, E and F to each represent hydrogen and fori to be zero. In compounds of particular interest the values for c, dand f are each 3 (as in bis (3-aminopropyl)-1,3-propanediamine) or,respectively 3, 4 and 3 (as in spermine).

Although generally the substituents G and J each represent hydrogen, Gand J may represent certain organic groups as indicated and particularlyeach of G and J may independently represent C₁ -C₄ alkyl or C₃ -C₆cycloalkyl or together may represent a C₂ -C₆ alkylene group, forexample a tetramethylene, pentamethylene or hexamethylene group, or agroup --CH₂ CH₂ --O--CH₂ CH₂ -- (to form a morpholine ring system).

Compounds of formula I which are of particular interest include thosewith the following formulae: ##STR3##

Acid addition salts of compounds of formula (I) in accordance with theinvention are preferably pharmaceutically acceptable although other acidaddition salts are within the scope of the invention. Suitable salts arethose derived from, for example, the following acids: hydrochloric,hydrobromic, sulphuric, nitric, isethionic, phosphoric, maleic,salicylic, p-toluenesulphonic, tartaric, citric, lactobionic, formic,malonic, pantothenic, succinic, naphthalene-2-sulphonic,benzenesulphonic, methanesulphonic and ethanesulphonic. The preferredsalts in terms of pharmaceutical acceptability are the ethanesulphonicacid salts.

Such salts may be prepared from the free-base by treatment with the acidsuitably in a polar solvent such as water and if necessary with theapplication of heat.

The compounds of the present invention may be readily produced byreaction between an acid and an amine moiety.

Accordingly to a further aspect of the present invention a process forthe production of a compound of formula I comprises reacting an acid offormula II or esterifiable derivative thereof e.g. an acid halide##STR4## with an amine or of formula III or acid addition salt thereof:

    NH.sub.2 (CH.sub.2).sub.c N(D)(CH.sub.2).sub.d N(E)(CH.sub.2).sub.f [N(F)(CH.sub.2).sub.h ].sub.i NGJ,                        III

in which formulae the symbols have the hereinbefore given meanings.

In general the reaction is conducted in an organic solvent such as1,2-dimethoxyethane or tetrahydrofuran or dioxan in the presence of acoupling reagent such as a carbodiimide e.g. dicyclohexylcarbodiimide oranother carbodiimide e.g.1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulphonate(morpho CDI, Aldrich), or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride or methiodide (water soluble reagents, EDC, Aldrich), or1,3-diisopropylcarbodiimide. The product may be readily purified bychromatography and/or lyophilisation.

The acid (or esterifiable derivative) thereof may, for example, bebenzoic, phenylacetic, phenylpropanoic, or cinnamic or substitutedderivatives e.g. hydroxyphenylglycine, dopa, tyrosine,hydroxyphenylpyruvic-, hydroxyphenylacetic- and hydroxymandelic acid.

The present invention further includes within its scope a compositionfor the treatment of psychoses, senile dementia, ischaemia, stroke,hypoxia, aneurysm, epilepsy, Parkinson's disease, Alzheimer's disease,Huntington's chorea, and related syndromes and neurological disorderswhich comprises a compound of formula I together with a pharmaceuticallyacceptable diluent or carrier.

The compounds of the present invention may be formulated by standard ornovel techniques to give, if desired, unit dosage forms. Although thedosage for a particular clinical application must be ascertained bytrial, for general guidance appropriate intravenous dosages usually liein the range 5-500 μg/kg but exceptionally above this i.e. up to 1 mg/kgor, rarely, up to 5 mg/kg. Oral dosages usually lie in the range 1-100mg/kg.

The present invention also includes within its scope a method oftreating a patient suffering from a psychotic disorder, senile dementia,ischaemia, stroke, hypoxia, aneurysm, epilepsy, Parkinson's disease,Alzheimer's disease, Huntington's chorea or a related syndrome orneurological disorder, in which the patient is administered an amount ofa compound of formula I effective to reduce, remove or prevent symptomsof the disorder.

The compound may be administered orally, parenterally (includingsubcutaneously, intramuscularly and intravenously). The administrationwill generally be carried out repetitively at intervals, for exampleonce or several times a day.

The amount of compounds of formula (I), as hereinbefore defined inaccordance with the invention, which is required in order to beeffective for treating mammals will, of course, vary and is ultimatelyat the discretion of the medical or veterinary practitioner treating themammal in each particular case. The factors to be considered by such apractitioner, e.g. a physician, include the route of administration andpharmaceutical formulation; the mammal's body weight, surface area, ageand general condition; and the particular salt to be administered.

The present invention further includes within its scope a compound offormula I hereinbefore described for use in therapy.

The invention is illustrated by the following Examples:

GENERAL PROCEDURE FOR THE PREPARATION OF MONOACYL SPERMINES

To a solution of the required carboxylic acid in an organic solvent suchas 1,2-dimethoxyethane or dichloromethane an activating agent e.g.dicyclohexyl carbodiimide was added. The polyamine was introduced inslight excess (two-four fold) in order to increase the yield of thedesired monoacylated product. The product was isolated by chromatographyover Kieselgel 60 (230-400 mesh). The final product was always solublein water and was therefore purified by lyophilisation as a final step.

Locust Test Method and Rationale

The compounds were assayed for biological activity on the isolated,retractor unguis nerve-muscle preparation of the locust Schistocercagregaria (Usherwood, P.N.R and Machili, P. (1968) J. Exp. Biol., 49:341.). Isolated muscle preparations were bathed continuously in standardlocust saline in a bath of volume 250 μl. Twitch tension recordings ofthe muscle were made to electrical stimulation of its motor innervationsupramaximal voltage, frequency 0.1 Hz).

The locust isolated retractor unguis nerve-muscle is a preparation withwell characterised muscle receptors belonging to thequisqualate-sensitive class (Boden, P. et al., (1986) Brain Res., 385:205.). This preparation has previously been employed to identifyantogonists of excitatory amino acid receptors, (e.g. Bateman, A. etal., (1985) Brain Res., 339: 237 and Budd, T. et al., (1988) Brain Res.,448: 30.). Excitatory amino acid receptors are also believed to be ofconsiderable importance in the mammal and for therapeutic applications.

EXAMPLE 1 N-(2-Hydroxyphenylacetyl)-spermine

To a solution of 2-hydroxyphenylacetic acid (50 mg, 0.33 mMol) in1,2-dimethoxyethane (DME) (1 ml) was added a solution of dicyclohexylcarbodiimide (71 mg, 0.34 mMol, 1.05 equivalents) in DME (1 ml) in oneportion of 25° C. The colourless mixture was allowed to stand at 25° C.for 3 h during which time a voluminous white precipitate of the urea wasformed. This precipitate was then removed by filtration and the residuewashed with DME (2×1 ml). The filtrate and the washings were combinedand a solution of spermine (351 mg, 1.73 mMol, 5.3 equivalents) inDME/dimethylformamide (2 ml, 1:1) was added in one portion. Thecolourless, homogeneous solution was sealed under an atmosphere ofnitrogen and allowed to stand at 25° C. for 48 h. The solution wasconcentrated in vacuo (water aspirator) and then the residue was appliedto a column of silica gel (Kieselgel 60, 230-400 mesh). The product waseluted with dichloromethane/methanol/0.880 ammonia solution (4:2:1). Thedesired amide was eluted in fractions 9-14 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.25, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a colourlessoil. This oil was dissolved in methanol (2 ml), filtered and thesolution was then concentrated in vacuo (51 mg, 46%). The residue wasdissolved in distilled water (2 ml) and lyophilised which gave the amideas a colourless, viscous oil (48 mg) whose spectroscopic data include:UV_(max) 271 (e_(max) 1 000) and 289 sh nm; pmr (90 MHz, ² H₂ O) 1.5-1.9(m, 8H, 4×CH₂ --CH₂), 2.6-2.9 (m, 10H, 5×CH₂ --N), 3.25 (t, J=7, 2H,CON² H--CH₂ --CH₂, 3.55 (s, 2H, Ar--CH₂ --CO), 6.55-6.85 (m, 2H, 2×ArH3,5), and 7.1-7.3 (m, 2H, 2×ArH 4,6)ppm; M+1 337 (FAB matrixm-nitrobenzyl alcohol) C₁₈ H₃₂ N₄ O₂ requires M⁺ 336).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced (1% inhibition) from control(100%) levels at the following concentrations:

    ______________________________________                                        3 × 10.sup.-8 M                                                                          10% inhibition of twitch                                     3 × 10.sup.-7 M                                                                          15%                                                          3 × 10.sup.-6 M                                                                          20%                                                          1.5 × 10.sup.-5 M                                                                        30%                                                          3 × 10.sup.-5 M                                                                          30%                                                          1.5 × 10.sup.-4 M                                                                        85%                                                          3 × 10.sup.-4 M                                                                          90%                                                          The IC.sub.50 = 6.2 × 10.sup.-5 M                                       ______________________________________                                    

EXAMPLE 2 N-(3-Hydroxyphenylacetyl)-spermine

To a solution of 3-hydroxyphenylacetic acid (50 mg, 0.33 mMol) in1,2-dimethoxyethane (DME) (1 ml) was added a solution of dicyclohexylcarbodiimide (75 mg, 0.36 mMol, 1.1 equivalents) in DME (1 ml) in oneportion of 25° C. The colourless mixture was allowed to stand at 25° C.for 3 h during which time a voluminous white precipitate of the urea wasformed. This precipitate was then removed by filtration and the residuewashed with DME (2×1 ml). The filtrate and the washings were combinedand a solution of spermine (167 mg, 0.83 mMol, 2.5 equivalents) inDME/dimethylformamide (2 ml, 1:1) was added in one portion. Thecolourless, homogeneous solution was sealed under an atmosphere ofnitrogen and allowed to stand at 25° C. for 24 h. The solution wasconcentrated in vacuo (water aspirator) and then the residue was appliedto a column of silica gel (Kieselgel 60, 230-400 mesh). The product waseluted with dichloromethane/methanol/0.880 ammonia solution (4:2:1). Thedesired amide was eluted in fractions 12-18 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.18, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a colourlessoil. This oil was dissolved in methanol (2 ml), filtered and thesolution was then concentrated in vacuo (36 mg, 33%). The residue wasdissolved in distilled water (2 ml) and lyophilised which gave the amideas a colourless, viscous oil (30 mg) whose spectroscopic data include:UV_(max) 274 sh, 278 (e_(max) 950), and 290 sh nm; pmr (90 MHz, ² H₂ O)1.2-2.0 (m, 8H, 4×CH₂ --CH₂), 2.5-2.9 (m, 10H, 5×CH₂ --N), 3.3 (t, J=7,2H, CON² H--CH₂ --CH₂), 3.5 (s, 2H, Ar--CH₂ --CO), 6.45-6.75 (m, 3H,3×ArH 2,4,6), and 7.0-7.3 (m, 1H, ArH 5)ppm: M+1 337 (FAB matrixm-nitrobenzyl alcohol) (C₁₈ H₃₂ N₄ O₂ requires M⁺ 336).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control (100%) levels atthe following concentrations:

    ______________________________________                                               3 × 10.sup.-8 M                                                                  30%                                                                  3 × 10.sup.-7 M                                                                  35%                                                           ______________________________________                                    

EXAMPLE 3 N-(4-Hydroxyphenylacetyl)-spermine

To a solution of 4-hydroxyphenylacetic acid (80 mg, 0.53 mMol) in1,2-dimethoxyethane (DME) (1 ml) was added a solution of dicyclohexylcarbodiimide (120 mg, 0.58 mMol, 1.1 equivalents) in DME (1 ml) in oneportion at 25° C. The colourless mixture was allowed to stand at 25° C.for 3 h during which time a voluminous white precipitate of the urea wasformed. This precipitate was then removed by filtration and the residuewashed with DME (2×1 ml). The filtrate and the washings were combinedand a solution of spermine (600 mg, 2.97 mMol, 5.6 equivalents) inDME/dimethylformamide (2 ml, 1:1) was added in one portion. Thecolourless, homogeneous solution was sealed under an atmosphere ofnitrogen and allowed to stand at 25° C. for 48 h. The solution wasconcentrated in vacuo (water spirator) and then the residue was appliedto a column of silica gel (Kieselgel 60, 230-400 mesh). The product waseluted with dichloromethane/methanol/0.880 ammonia solution (4:2:1). Thedesired amide was eluted in fractions 8-13 (10 ml fractions) and washomogeneous when monitored by tic on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.17, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a colourlessoil. This oil was dissolved in methanol (2 ml), filtered and thesolution was then concentrated in vacuo (65 mg, 37%). The residue wasdissolved in distilled water (2 ml) and lyophilised which gave the amideas a colourless, viscous oil (57 mg) whose spectroscopic data include:UV_(max) 220 (e_(max) 6 000) and 270 (e_(max) 1 300) nm: pmr (90 MHz, ²H₂ O) 1.2-2.0 (m, 8H, 4×CH₂ --CH ₂ --CH₂), 2.5-2.9 (m, 10H, 5×CH₂ --N),3.2 (t, J=7, 2H, CON² H--CH₂ --CH₂), 3.4 (s, 2H, Ar--CH₂ --CO), 6.7 (d,J=8, 2H, 2×ArH 3,5), and 7.1 (d, J=8, 2H, 2×ArH 2,6)ppm: M+1 337 (FABmatrix m-nitrobenzyl alcohol) (C₁₈ H₃₂ N₄ O₂ requires M+336).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control (100%) levels atthe following concentrations:

    ______________________________________                                        5.9 × 10.sup.-7 M                                                                         9%                                                          5.9 × 10.sup.-6 M                                                                        25%                                                          2.95 × 10.sup.-5 M                                                                       74%                                                          4.43 × 10.sup.-5 M                                                                       91%                                                          5.9 × 10.sup.-5 M                                                                        96%                                                          The IC.sub.50 = 8.7 × 10.sup.-6 M                                       ______________________________________                                    

EXAMPLE 4 N-(2-Hydroxyphenylpropanoyl)-spermine

To a solution of 2-hydroxyphenylpropanoic acid (61 mg, 0.37 mMol) in1,2-dimethoxyethane (DME) (1 ml) was added a solution ofdicyclohexylcarbodiimide (76 mg, 0.37 mMol) in DME (1 ml) in one portionat 25° C. The colourless mixture was allowed to stand at 25° C. for 3 hduring which time a voluminous white precipitate of the urea was formed.This precipitate was then removed by filtration and the residue washedwith DME (2×1 ml). The filtrate and the washings were combined and asolution of spermine (300 mg, 1.48 mMol, 4.0 equivalents) inDME/dimethylformamide (2 ml, 1:1) was added in one portion. Thecolourless, homogeneous solution was sealed under an atmosphere ofnitrogen and allowed to stand at 25° C. for 48 h. The solution wasconcentrated in vacuo (water aspirator) and the residue was then appliedto a column of silica gel (Kieselgel 60, 230-400 mesh). The product waseluted with dichloromethane/methanol/ 0.880 ammonia solution (4:2:1).The desired amide was eluted in fractions 7-11 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.33, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a colourlessoil.

This oil was dissolved in methanol (2 ml), filtered and the solution wasthen concentrated in vacuo (112 mg, 87%). The residue was dissolved indistilled water (2 ml) and lyophilised which gave the amide as acolourless, viscous oil (82 mg) whose spectroscopic data include: pmr(90 MHz, ² H₂ O) 1.3-2.1 (m, 8H, 4×CH₂ --CH₂), 2.5-3.1 (m, 14H, 5×CH₂--N and Ar--CH₂ --CH₂), 3.3 (t, J=7, 2H, CON² H--CH₂ --CH₂), 6.7-7.0 (m,2H, 2×ArH 3,5), and 7.1-7.4 (m, 2H, 2×ArH 4,6)ppm.

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electrcicallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control levels between thefollowing concentrations: 1×10⁻⁵ M and 9×10⁻⁵ M, such that anapproximately 50% reduction was obtained at 5×10⁻⁵ M.

EXAMPLE 5 N-(3-Hydroxyphenylpropanoyl)-spermine

To a solution of 3-hydroxyphenylpropanoic acid (167 mg, 1.01 mMol) indichloromethane (5 ml) was added dicyclohexylcarbodiimide (226 mg, 1.10mMol, 1.1 equivalents) in one portion at 25° C. The colourless mixturewas allowed to stand at 25° C. for 2 h during which time a voluminouswhite precipitate of the urea was formed. This precipitate was thenremoved by filtration and the residue washed with dichloromethane (1ml). The filtrate and the washings were combined and spermine (400 mg,1.98 mMol, 1.96 equivalents) was added in one portion. The colourless,homogeneous solution was sealed under an atmosphere of nitrogen andallowed to stand at 25° C. for 4 h. The solution was concentrated invacuo (water aspirator) and the residue was then applied to a column ofsilica gel (Kieselgel 60, 230-400 mesh). The product was eluted withdichloromethane/methanol/0.880 ammonia solution (4:2:1). The desiredamide was eluted in fractions 13-19 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.11, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a colourlessoil. This oil was dissolved in methanol (2 ml), filtered and thesolution was then concentrated in vacuo (105 mg, 30%). The residue wasdissolved in distilled water (2 ml) and lyophilised which gave the amideas a colourless glass (80 mg) whose spectroscopic data include: UV_(max)217 (e_(max) 2 500) and 272 (e_(max) 1 200)nm; pmr (90 MHz, ² H₂ O)1.5-2.0 (m, 8H, 4×CH₂ -- CH₂), 2.4-3.0 (m, 14H, 5×CH₂ --N and Ar--CH₂--CH₂), 3.2 (t, J=7, 2H, CON² H--CH₂ --CH₂), 6.55-6.75 (m, 3H, 3×ArH2,4,6), and 7.05-7.3 (m, H, ArH 5)ppm; M+1 351 (FAB matrix m-nitrobenzylalcohol) C₁₉ H₃₄ N₄ O₂ requires M⁺ 350).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schisocerca gregaria).Twich amplitude was reduced from control (100%) levels at the followingconcentrations:

    ______________________________________                                        1.08 × 10.sup.-7 M                                                                       10% inhibition of twitch                                     1.08 × 10.sup.-6 M                                                                       15%                                                          ______________________________________                                    

EXAMPLE 6 N-(4-Hydroxycinnamoyl)-spermine

To a solution of 4-hydroxycinnamic acid (82 mg, 0.50 mMol) in1,2-dimethoxyethane (DME) (1 ml) was added a solution ofdicyclohexylcarbodiimide (106 mg, 0.51 mMol, 1.03 equivalents) in DME (1ml) in one portion at 25° C. The colourless mixture was allowed to standat 25° C. for 3 h during which time a voluminous white precipitate ofthe urea was formed. This precipitate was then removed by filtration andthe residue washed with DME (2×1 ml). The filtrate and the washings werecombined and a solution of spermine (336 mg, 1.66 mMol, 3.3 equivalents)in DME/dimethylformamide (2 ml, 1:1) was added in one portion. Theyellow, homogeneous solution was sealed under an atmosphere of nitrogenand allowed to stand at 25° C. for 48 h. The solution was concentratedin vacuo (water aspirator) and the residue was then applied to a columnof silica gel (Kieselgel 60, 230-400 mesh). The product was eluted withdichloromethane/methanol/0.880 ammonia solution (4:2:1). The desiredamide was eluted in fractions 11-16 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.21, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a pale yellowoil.

This oil was dissolved in methanol (2 ml), filtered and the solution wasthen concentrated in vacuo (30 mg, 17%). The residue was dissolved indistilled water (2 ml) and lyophilised which gave the amide as acolourless, viscous oil (30 mg) whose spectroscopic data include:UV_(max) 224 293 and 312 sh nm; pmr (90 MHz, ² H₂ O) 1.4-2.0 (m, 8H,4×CH₂ --CH₂), 2.6-3.0 (m, 10H, 5×CH₂ --N), 3.35 (t, J=7, 2H, CON² H--CH₂--CH₂), 6.36 (d, J=16, 1H, CH═CH--CO) 6.6-6.9 (m, 2H, 2×ArH 3,5), and7.2-7.6 (m, 3H, 2×ArH 2,6 and Ar--CH═CH)ppm; M+1 349 (FAB matrixm-nitrobenzyl alcohol) C₁₉ H₃₂ N₄ O₂ requires M+ 348).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control (100%) levels atthe following concentrations:

    ______________________________________                                        10.sup.-10 M.sup.                                                                              4%                                                           10.sup.-9 M     18%                                                           10.sup.-8 M     24%                                                           10.sup.-7 M     33%                                                           10.sup.-6 M     56%                                                           10.sup.-5 M     70%                                                           The IC.sub.50 = 6 × 10.sup.-5 M                                         ______________________________________                                    

EXAMPLE 7 N-(2,4-Dihydroxyphenylacetyl)-spermine

To a solution of 2,4-dibenzyloxyphenylacetic acid N-hydroxysuccinimideester (93 mg, 0.22 mMol) in dichloromethane (3 ml) was added spermine(112 mg, 0.55 mMol, 2.5 equivalents)) in one portion. The colourless,homogeneous solution was sealed under an atmosphere of nitrogen andallowed to stand at 25° C. for 24 h. The solution was concentrated invacuo (water aspirator) and the residue was then applied to a column ofsilica gel (Kieselgel 60, 230-400 mesh). The product was eluted withdichloromethane/methanol/-0.880 ammonia solution (8:4:1). The desiredamide was eluted in fractions 10-15 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.22, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a pale yellowgum.

This gum was dissolved in methanol (2 ml), filtered and the solution wasthen concentrated in vacuo (50 mg, 45%). The residue was dissolved indistilled water (2 ml) and lyophilised which gave the amide as a whitesolid (43 mg). The amide was dissolved in water (2 ml) and aqueoushydrochloric acid (2M, 0.5 ml) and palladium on carbon (10%, 11 mg) werethen added. The mixture was stirred and hydrogenated at 1 atm and 25° C.for 24 h. The catalyst was removed by filtration through a pad ofKieselguhr, eluted with water. The clear solution was then lyophilisedwhich gave the desired amide trihydrochloride (34 mg) as a colourlessglass whose spectroscopic data include: UV_(max) 293 (e_(max) 1 500)nmat pH=11; M+1 353 (FAB matrix m-nitrobenzyl alcohol) C₁₈ H₃₂ N₄ O₃requires M⁺ 352).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using electrically stimulatedthe retractor unguis muscle of the locust (Schistocerca gregaria).Twitch amplitude was reduced from control levels between the followingconcentrations: 1×10⁻⁵ M and 9×10⁻⁵ M, such that an approximately 50%reduction was obtained at 5×10⁻⁵ M.

EXAMPLE 8 N-(3,4-Dihydroxyphenylacetyl)-spermine

To a solution of 3,4-dibenzyloxyphenylacetic acid N-hydroxysuccinimideester (115 mg, 0.26 mMol) in dichloromethane (5 ml) was added spermine(128 mg, 0.63 mMol, 2.45 equivalents) in one portion. The colourless,homogeneous solution was sealed under an atmosphere of nitrogen andallowed to stand at 25° C. for 22 h. The solution was concentrated invacuo (water aspirator) and the residue was then applied to a column ofsilica gel (Kieselgel 60, 230-400 mesh). The product was eluted withdichloromethane/methanol/0.880 ammonia solution (8:4:1). The desiredamide was eluted in fractions 18-26 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,8:4:1), R_(f) =0.16, chromatogram visualised by inspection under UVlight (254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a pale yellowgum.

This gum was dissolved in methanol (2 ml), filtered and the solution wasthen concentrated in vacuo (48 mg, 35%). The residue was dissolved indistilled water (2 ml) and lyophilised which gave the amide as acolourless, viscous oil (46 mg). The amide was dissolved in water (2 ml)and aqueous hydrochloric acid (2M, 0.5 ml) and palladium on carbon (10%,12 mg) were then added. The mixture was stirred and hydrogenated at 1atm and 25° C. for 24 h. The catalyst was removed by filtration througha pad of Kieselguhr, eluted with water. The clear solution was thenlyophilised which gave the desired amide trihydrochloride as acolourless glass (32 mg) whose spectroscopic data include: UV_(max) 2700) and 291 (e_(max) 2 500)nm at pH=11.

The potency of this monoacylated spermine as a non-competitiveantagonist of the complex formed by the invertebrate L-quisqualatesub-type L-glutamate receptor and its ion channel was assayed using theelectrically stimulated retractor unguis muscle of the locust(Schistocerca gregaria). Twitch amplitude was reduced from controllevels between the following concentrations: 1×10⁻⁴ M and 9×10⁻⁴ M, suchthat an approximately 50% reduction was obtained at 5×10⁻⁴ M.

EXAMPLE 9 N-(3,5-Dihydroxyphenylacetyl)-spermine

To a solution of 3,5-dibenzyloxyphenylacetic acid (86 mg, 0.247 mMol) indichloromethane (1 ml) was added a solution of dicyclohexylcarbodiimide(55 mg, 0.27 mMol 1.08 equivalents) in dichloromethane (1 ml) in oneportion at 25° C. The colourless mixture was allowed to stand at 25° C.for 1.5 h during which time a voluminous white precipitate of the ureawas formed. This precipitate was then removed by filtration and theresidue washed with dichloromethane (2×1 ml). The filtrate and thewashings were combined and a solution of spermine (150 mg, 0.74 mMol,3.0 equivalents) in dichloromethane (1 ml) was added in one portion. Thecolourless, homogeneous solution was sealed under an atmosphere ofnitrogen and allowed to stand at 25° C. for 4 h. The solution wasconcentrated in vacuo (water aspirator) and the residue was then appliedto a column of silica gel (Kieselgel 60, 230-400 mesh). The product waseluted with dichloromethane/methanol/0.800 ammonia solution (8:4:1). Thedesired amide was eluted in fractions 15-18 (10 ml fractions) and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1) R_(f) =0.11, chromatogram visualised by inspection under UV light(254 nm) and by the production of a pink-purple stain with 0.3%ninhydrin in solution in acidic alcohol. The fractions containing theproduct were combined and concentrated in vacuo to yield a pale yellowgum.

This gum was dissolved in methanol (2 ml), filtered and the solution wasthen concentrated in vacuo (29 mg, 22%). The residue was dissolved indistilled water (2 ml) and lyophilised which gave the amide as a paleyellow solid (29 mg). The amide was dissolved in water (2 ml) andaqueous hydrochloric acid (2M, 0.5 ml) and palladium on carbon (10%, 11mg) were then added. The mixture was stirred and hydrogenated at 1 atmand 25° C. for 22 h. The catalyst was removed by filtration through apad of kieselguhr, eluted with water. The clear solution was thenlyophilised which gave the desired amide trihydrochloride (24 mg) as acolourless glass whose spectroscopic data include:UV_(max) 290 (e_(max)1 300) nm at pH=11; M+1 353 (FAB matrix m-nitrobenzyl alcohol) C₁₈ H₃₂N₄ O₃ requires M⁺ 352).

The potency of this monoacylated spermine as an antagonist of thecomplex formed by the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control levels between thefollowing concentrations:

    ______________________________________                                               1 07 × 10.sup.-6 M                                                                25%                                                                 1.07 × 10.sup.-5 M                                                                45%                                                          ______________________________________                                    

EXAMPLES 10-21

In the present Examples, compounds of formula I were prepared byfollowing the above General Procedure and adapting the methods describedin Examples 1 to 7. The compounds prepared together with an indicationof their potency as L-glutamate antagonists when tested in the locustleg retractor muscle pharmacological screen follow:

EXAMPLE 10 N-2-Hydroxybenzoyl)-spermine

According to the General Procedure using 2-hydroxybenzoic acid (138 mg,1.00 mMol). dicyclohexylcarbodiimide (226 mg, 1.10 mMol), and spermine(400 mg, 1.98 mMol) in dichloromethane (6 ml), activation during 3 h andcoupling over 4 h. The product was eluted over silica gel withdichloromethane/methanol/0.880 ammonia solution (4:2:1). The desiredamide was in fractions 8-15 and was homogeneous when monitored by tlc onsilica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f) =0.23, (98 mg, 29%),lyophilisation gave 64 mg of a colorless, viscous oil whosespectroscopic data include:pmr (90 MHz, ² H₂ O) 1.5-1.9 (m, 8H, 4×CH₂--CH₂, 2.6-2.9 (m, 10H, 5×CH₂ --N), 3.25 (t, J=7, 2H, CON² H--CH₂ --CH₂,6.55-6.85 (m, 2H, 2×ArH 3,5), and 7.1-7.3 (m, 2H, 2×ArH 4,6) ppm; M+1323 (FAB matrix m-nitrobenzyl alcohol and sodium chloride) C₁₇ H₃₀ N₄ O₂requires M⁺ 322).

The potency of this monoacylated spermine as an antagonist of theinvertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control level at 1.09×10⁻⁵ M by approximately 25%.

EXAMPLE 11 N-(3-Hydroxybenzoyl)-spermine

According to the General Procedure using 3-Hydroxybenzoic acid (138 mg,1.00 mMol), dicyclohexylcarbodiimide (226 mg, 1.10 mMol), and spermine(400 mg, 1.98 mMol) in dichloromethane (6 ml), activation during 3 h andcoupling over 17 h. The product was eluted over silica gel withdichloromethane/methanol/0.880 ammonia solution (4:2:1). The desiredamide was in fractions 8-13 was homogeneous when monitored by tlc onsilica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f) =0.29, (76 mg, 24%),lyophilisation gave 52 mg of a colorless, viscous oil whosespectroscopic data include:pmr (90 MHz, ² H₂ O) 1.4-1.9 (m, 8H, 4×CH₂--CH₂), 2.5-2.9 (m, 10H, 5×CH₂ --N), 3.25 (t, J=7, 2H, CON² H--CH₂--CH₂), 6.5-6.8 (m, 3H, 3× ArH 2,4,6), and 7.0-7.3 (m, 1H, ArH 5) ppm;M+1 323 (FAB matrix m-nitrobenzyl alcohol and sodium chloride) C₁₇ H₃₀N₄ O₂ requires M⁺ 322).

The potency of this monoacylated spermine as an antagonist invertebrateL-quisqualate sub-type L-glutamate receptor and its ion channel wasassayed using the electrically stimulated retractor unguis muscle of thelocust (Schistocerca gregaria). Twitch amplitude was reduced fromcontrol levels at 1.02×10⁻⁵ M by approximately 40%.

EXAMPLE 12 N-(4-Hydroxybenzoyl)-spermine

According to the General Procedure using 4-hydroxybenzoic acid (80 mg,0.58 mMol), dicyclohexylcarbodiimide (125 mg, 0.61 mMol), and spermine(430 mg, 2.13 mMol) in DME/DMF (4 ml, 3:1), activation during 3 h andcoupling over 48 h. The product was eluted over silica gel withdichloromethane/methanol/0.880 ammonia solution (2:2:1). The desiredamide was in fractions 8-12 and was homogeneous when monitored by tic onsilica (CH₂ Cl₂ /MeOH/NH₄ OH, 2:2:1), R_(f) =0.29, (23 mg, 12%),lyophilisation gave 7 mg of a colourless, viscous oil whosespectroscopic data include: pmr (90 MHz, ² H₂ O) 1.4-1.9 (m, 8H, 4×CH₂--CH₂), 2.5-2.9 (m, 10H, 5×CH₂ --N), 3.25 (t, J=7, 2H, CON² H--CH₂--CH₂) 6.6-6.9 (m, 2H, 2×ArH 3,5), and 7.0-7.3 (m, 2H, 2×ArH2,6)ppm; M+1323 (FAB matrix m-nitrobenzyl alcohol) C₁₇ H₃₀ N₄ O₂ requires M⁺ 322).

The potency of this monoacylated spermine as an antagonist of theinvertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control levels: between 10⁻⁵ M and 10⁻⁴ M such that anapproximately 50% reduction was obtained at 5×10⁻⁵ M.

EXAMPLE 13 N-(3-Hydroxycinnamoyl)-spermine

According to the General Procedure using 3-hydroxycinnamic acid (160 mg,0.97 mMol), dicyclohexylcarbodiimide (226 mg, 1.10 mMol), and spermine(400 mg, 1.98 mMol) in dichloromethane (6 ml), activation during 2 h andcoupling over 21 h. The product was eluted over silica gel withdichloromethane/methanol/0.880 ammonia solution (4:2:1). The desiredamide was in fractions 15-22 and was homogeneous when monitored by tlcon silica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f) =0.22, (108 mg, 32%),lyophilisation gave 90 mg of a yellow viscous gum whose spectroscopicdata include: UV_(max) 213 (e_(max) 3 500), 230sh and 375 (e_(max) 3800) nm; pmr (90 MHz, ² H₂ O) 1.4-2.0 (m, 8H, 4×CH₂ --CH₂), 2.6-3.0 (m,10H, 5×CH₂ --N), 3.35 (t, J=7, 2H, CON² H--CH₂ --CH₂), 6.4 (d, J=16, 1H,CH═CH--CO), 3.55 (s, 2H, Ar--CH₂ --CO), 6.6-6.9 (m, 3H, 3×ArH 2,4,6),and 7.2-7.6 (m, 2H, ArH 5 and ArCH═CH)ppm; M+1 349 (FAB matrixm-nitrobenzyl alcohol) C₁₉ H₃₂ N₄ O₂ requires M⁺ 348).

The potency of this monoacylated spermine as an antonist of theinvertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control levels at 9.2×10⁻⁷ M by approximately 25%.

EXAMPLE 14 N-(3-Fluoro-4-hydroxyphenylacetyl)-spermine.

According to the General Procedure using 3-fluoro-4-hydroxyphenylaceticacid (85 mg, 0.50 mMol), dicyclohexylcarbodiimide (106 mg, 0.31 mMol),and spermine (512 mg, 2.53 mMol) in DME/DMF (4 ml, 3:1), activationduring 2 h and coupling over 48 h. The product was eluted over silicagel with dichloromethane/methanol/0.880 ammonia solution (4:2:1). Thedesired amide was in fractions 12-19 and was homogeneous when monitoredby tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f) =0.19, (39 mg,22%), lyophilisation gave 30 mg of a colourless, viscous oil whosespectroscopic data include: pmr (90 MHz, ² H₂ O) 1.6-2.2 (m, 8H, 4×CH₂--CH₂), 2.85-3.2 (m, 10H, 5×CH₂ --N), 3.4 (t, J=7, 2H, CON² H--CH₂--CH₂), 3.6 (s, 2H, Ar--CH₂ --CO), 6.8-7.1 (m, 2H, 2×ArH 5,6), and 7.1(d, 3 J H--F=11, H, ArH 2)ppm.

The potency of this monoacylated spermine as an antagonist of theinvertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control levels between 10⁻⁴ M and 10⁻³ M, such that anapproximately 50% reduction was obtained at 5×10⁻⁴ M.

EXAMPLE 15 N-(2-Methoxyphenylacetyl)-spermine

According to the General Procedure using 2-methoxyphenylacetic acid (81mg. 0.49 mMol), dicyclohexylcarbodiimide (53 mg, 0.26 mMol), andspermine (201 mg, 0.99 mMol) in DME (4 ml), activation during 2.5 h andcoupling over 48 h. The produce was eluted over silica gel with thelower layer of dichloromethane/methanol/0.880 ammonia solution (2:1:1).The desired amide was in fractions 6-10 and was homogeneous whenmonitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH lower layer, 2:1:1),R_(f) =0.25, (28 mg, 31%), lyophilisation gave 16 mg of a colourless,viscous oil whose spectroscopic data include: pmr (90 MHz, ² H₂ O)1.35-1.85 (m, 8H, 4×CH₂ --CH₂), 2.4-2.9 (m, 10H, 5×CH₂ --N), 3.25 (t,J=7, 2H, CON² H--CH₂ --CH₂), 3.5 (s, 2H, Ar--CH₂ --CO), 3.83(s, 3H,OCH₃), 6.75-7.05 (m, 2H, 2×ArH 3,5), and 7.15-7.35 (m, 2H, 2×ArH4,6)ppm.

The potency of this monoacylated spermine as a non-competitiveantagonist of the invertebrate L-quisqualate sub-type L-glutamatereceptor and its ion channel was assayed using the electricallystimulated retractor unguis muscle of the locust (Schistocercagregaria). Twitch amplitude was reduced from control (100%) levelsbetween 1×10⁻⁴ M and 1×10⁻³ M, such that an approximately 50% reductionwas obtained at 5×10⁻⁴ M.

EXAMPLE 16 N-(4-Methoxyphenylacetyl)-spermine

According to the General Procedure using 4-methoxyphenylacetic acid (83mg, 0.50 mMol), dicyclohexylcarbodiimide (56 mg, 0.27 mMol), andspermine (201 mg, 0.99 mMol) in DME (4 ml), activation during 2.5 h andcoupling over 48 h. The product was eluted over silica gel with thelower layer of dichloromethane/methanol/0.880 ammonia solution (4:1:1).The desired amide was in fractions 16-25 and was homogeneous whenmonitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH lower layer, 4:1:1),R_(f) =0.07, (30 mg, 32%), lyophilisation gave 21 mg of a colourless,viscous oil whose spectroscopic data include: pmr (90 MHz, ² H₂ O)1.4-1.8 (m, 8H, 4×CH₂ --CH₂), 2.4-2.9 (m, 10H, 5×CH₂ --N), 3.3 (t, J=7,2H, CON² H--CH₂ --CH₂), 3.5 (s, 2H, Ar--CH₂ --CO), 3.83 (s, 3H, OCH₃),6.8-6.95 (m, 2H, 2×ArH 3,5), and 7.1-7.3 (m, 2H, 2×ArH 2,6)ppm.

The potency of this monoacylated spermine as an antagonist of theinvertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control (100%) levels between 1×10⁻⁴ M and 1×10⁻³ M, suchthat an approximately 50% reduction was obtained at 5×10⁻⁴ M.

EXAMPLE 17N-(2-Methoxyphenylacetyl)-N',N"-bis(3-aminopropyl)-1,3-propanediamine

According to the General Procedure using 2-methoxyphenylacetic acid (80mg, 0.48 mMol), dicyclohexylcarbodiimide (106 mg, 0.51 mMol), andbis(3-aminopropyl)-1,3-propanediamine (452 mg, 2.40 mMol) in DME (4 ml,activation during 2 h and coupling over 48 h. The product was elutedover silica gel with the lower layer of dichloromethane/methanol/0.880ammonia solution (2:1:1). The desired amide was in fractions 4-7 and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH lowerlayer, 2:1:1), R_(f) =0.23, (55 mg, 34%) lyophilisation gave 54 mg of acolourless, viscous oil whose spectroscopic data include: pmr (90 MHz,C² HCl₃) 0.9-1.4 (m, 4H, 4×NH), 1.3-2.0 (m, 6H, 3×CH₂ --CH₂), 2.3-2.9(m, 10H, 5×CH₂ --N), 3.22 (app. q, J=7, 2H, CONH--CH₂ --CH₂), 3.5 (s,2H, Ar--CH₂ --CO), 3.8(s, 3H, OCH₃), 6.35(br t, J=7, CONH--CH₂), 6.7-7.0(m, 2H, 2×ArH 3,5), and 7.1-7.35 (m, 2H, 2×ArH 4,6)ppm.

The potency of this compound as an antagonist of the invertebrateL-quisqualate sub-type L-glutamate receptor and its ion channel wasassayed using the electrically stimulated retractor unguis muscle of thelocust (Schistocerca gregaria). Twitch amplitude was reduced fromcontrol (100%) levels between 1×10⁻⁴ M and 9×10⁻³ M, such that anapproximately 50% reduction was obtained at 9×10⁻⁴ M.

EXAMPLE 18N-(4-Methoxyphenylacetyl)-N',N"-bis(3-aminopropyl)-1,3-propanediamine

According to the General Procedure using 4-methoxyphenylacetic acid (80mg, 0.48 mMol), dicyclohexylcarbodiimide (106 mg, 0.51 mMol), andbis(3-aminopropyl)-1,3-propanediamine (464 mg, 2.46 mMol) in DME (6 ml),activation during 2 h and coupling over 48 h. The product was elutedover silica gel with the lower layer of dichloromethane/methanol/0.880ammonia solution (2:1:1). The desired amide was in fractions 4-8 and washomogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH lowerlayer, 2:1:1), R_(f) =0.30, (35 mg, 22%) lyophilisation gave 26 mg of acolourless, viscous oil whose spectroscopic data include: pmr (90 MHz,C² HCl₃) 1.0-1.4 (m, 4H, 4×NH), 1.3-2.0 (m. 6H, 3×CH₂ --CH₂), 2.2-2.9(m, 10H, 5×CH₂ --N, 3.25 (app. q, J=7, 2H, CONH--CH₂ --CH₂), 3.42 (s,2H, Ar--CH₂ --CO), 3.76 (s, 3H, OCH₃), 6.63 (br t, J=7, CONH--CH₂), 6.8(d, J=8, 2H, 2×ArH3,5), and 7.15 (d, J=8, 2H, 2×ArH 2,6)ppm.

The potency of this compound as an antagonist of the complex formed bythe invertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control (100%) levels between 1×10⁻⁴ M and 9×10⁻³ M, suchthat an approximately 50% reduction was obtained at 9×10⁻⁴ M.

EXAMPLE 19N-(2-Hydroxyphenylacetyl)-N',N"-bis(3-aminopropyl)-1,3-propanediamine

According to the General Procedure using 2-hydroxyphenylacetic acid (160mg, 1.05 mMol), dicyclohexylcarbodiimide (230 mg, 1.11 mMol), andbis(3-aminopropyl)-1,3-propanediamine (806 mg, 4.28 mMol) in DME (6 ml),activation during 4.5 h and coupling over 24 h. The product was elutedover silica gel with dichloromethane/- methanol/0.880 ammonia solution(4:2:1). The desired amide was in fractions 8-20 and was homogeneouswhen monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f)=0.11, (235 mg, 69%), lyophilisation gave 188 mg of a colourless,viscous oil whose spectroscopic data include: pmr (90 MHz, C² HCl₃)1.4-1.8 (m, 6H, 3×CH₂ --CH₂), 2.3-2.85 (m, 10H, 5×CH₂ --N), 3.2 (t, J=7,2H, CON² H--CH₂ --CH₂), 3.5 (s, 2H, Ar--CH₂ --CO), 3.9 (br s, 6H,5×NHand OH) 6.65-6.9 (m, 2H, 2×ArH 3,5), and 6.95-7.2 (m, 2H, 2×ArH4,6)ppm.

The potency of this compound as an antagonist of the complex formed bythe invertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control (100%) levels between 1×10⁻⁴ M and 9×10⁻³ M, suchthat an approximately 50% reduction was obtained at 9×10⁻⁴ M.

EXAMPLE 20N-(3-Hydroxyphenylacetyl)-N',N"-bis(3-aminopropyl)-1,3-propanediamine

According to the General Procedure using 3-hydroxyphenylacetic acid (150mg, 0.98 mMol), dicyclohexylcarbodiimide (226 mg, 1.10 mMol), andbis(3-aminopropyl)-1,3-propanediamine (370 mg, 1.97 mMol) indichloromethane (6 ml), activation during 2 h and coupling over 3 h. Theproduct was eluted over silica gel with dichloromethane/methanol/0.880ammonia solution (4:2:1). The desired amide was in fractions 13-21 andwas homogeneous when monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH,4:2:1), R_(f) =0.27, (97 mg, 31%), lyophilisation gave 79 mg of acolourless, viscous oil whose spectroscopic data include: pmr (90 MHz, ²H₂ O) 1.4-2.0 (m, 6H, 3×CH₂ --CH₂), 2.3-2.9 (m, 10H, 5×CH₂ --N), 3.25(t, J=7, 2H, CON² H--CH₂ --CH₂), 3.5 (s, 2H, Ar--CH₂ --CO), 6.55-6.75(m, 3H, 3×ArH 2,4,6), and 7.05-7.3 (m, ArH 5)ppm.

The potency of this compound as an antagonist of the complex formed bythe invertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control (100%) levels at 1.04×10⁻⁴ M by approximately 60%.

EXAMPLE 21N-(4-Hydroxyphenylacetyl)-N',N"-bis(3-aminopropyl)-1,3-propanediamine

According to the General Procedure using 4-hydroxyphenylacetic acid (160mg, 1.05 mMol), dicyclohexylcarbodiimide (231 mg, 1.12 mMol), andbis(3-aminopropyl)-1,3-propanediamine (806 mg, 4.28 mMol) in DME (6 ml),activation during 4.5 h and coupling over 48 h. The product was elutedover silica gel with dichloromethane/methanol/0.880 ammonia solution(4:2:1). The desired amide was in fractions 11-20 and was homogeneouswhen monitored by tlc on silica (CH₂ Cl₂ /MeOH/NH₄ OH, 4:2:1), R_(f)=0.13, (88 mg 26%), lyophilisation gave 74 mg of a colorless gum whosespectroscopic data include: pmr (90 MHz, ² H₂ O) (1.4-2.0 (m, 6H, 3×CH₂--CH₂), 2.4-2.9 (m, 10H, 5×CH₂ --N), 3.18 (t, J=7, 2H, CON² H--CH₂--CH₂), 3.4 (s, 2 H, Ar--CH₂ --CO), 6.68(d, J=8, 2H, 2×ArH 3,5), and7.05(d, J=8, 2H, 2×ArH 2,6)ppm.

The potency of this compound as an antagonist of the complex formed bythe invertebrate L-quisqualate sub-type L-glutamate receptor and its ionchannel was assayed using the electrically stimulated retractor unguismuscle of the locust (Schistocerca gregaria). Twitch amplitude wasreduced from control (100%) levels between 1×10⁻⁴ M and 9×10⁻³ M, suchthat an approximately 50% reduction was obtained at 9×10⁻⁴ M, and onapproximately 30% reduction at 1×10⁻⁴ M.

EVALUATION ON A MAMMALIAN NEURONAL PREPARATION Method

Coronal slices of cerebral cortex were cut with a Vibratome from thebrains of freshly killed Wistar rats. Wedges of tissue were cut fromeither side of the mid-line to include the cortex and corpus callosum.The wedges were mounted in a grease-gap apparatus such that the cortexprotruded into one compartment and the corpus callosum into the othercompartment of a two-compartment bath, (Harrison N. L. & Simmonds M. A.(1985). Br. J. Pharmacol 84: 381-391.). Each compartment could beseparately perfused with Krebs bicarbonate medium pre-gassed with 95% 0₂/5% CO₂ at room temperature. The volume of each compartment was 1.8 mland the perfusion rate was 2 ml/min. The compartment containing thecortical part of the wedge was also gassed directly so that drugs couldbe injected into the bath with the perfusion pump stopped. This varianton the published method was adopted to minimise the amount of drugrequired. The potential difference between the two compartments wasrecorded continously via Ag/AgCl electrodes.

The polyamine compounds were tested for their ability to antagoniseeither the quizqualate receptor agonist(RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (TocrisNeuramin) or the NMDA receptor agonist N-methyl-D-aspartate (Sigma). Thepolyamine compounds were supplied as freeze-dried aliquots and, for eachexperiment, one of these was dissolved in sufficient distilled water toproduce a 10 mM stock solution. To permit optimal responses to NMDA,Mg²⁺ was omitted from the medium. Since this generally results inepileptiform discharges in the wedges, (Horne A. L., Harrison N. L.,Turner J. P. and Simmonds M. A. (1986) Eur. J. Pharmacol 122: 231-238)tetrodotoxin (Sigma) was used to suppress such activity and wasinitially added to the bath to a concentration of 2.5 μM for 5 min. andthereafter was incorporated into the perfusion medium at a concentrationof 0.1 μM.

The experimental protocol was to perfuse the tissue initially to obtaina steady baseline. The perfusion was then stopped and an aliquot of a 1mM stock solution of either AMPA or NMDA was injected into the corticalcompartment to give a concentration of 2.5 or 5 μM. Once thedepolarisation response had reached its peak (usually within 3 min.),the perfusion was resumed to wash out the drug and return the potentialto baseline. This procedure was repeated 3 or 4 times to achieveconsistent responses. With the perfusion again stopped, the polyaminewas injected into the cortical compartment to a concentration of 1, 10or 100 μM, using stock solutions of 0.1, 1 or 10 mM, respectively. After20 min, a dose of AMPA or NMDA was injected, as before, followed by theresumption of perfusion after the peak of the response. Repeatedinjections of AMPA or NMDA were made without further addition of thepolyamine. In control experiments, no polyamine was injected.

COMPOUND OF EXAMPLE 3

The potency of this monoacylated spermine was tested as an antagonist ofNMDA- and AMPA- induced response in a rat brain slice model.Electrophysiologically recorded depolarisation responses were reducedfrom control levels (% inhibition) at the following concentrations:

    ______________________________________                                                     AMPA  NMDA                                                       ______________________________________                                        10.sup.-6 M     0%     29%                                                    10.sup.-5 M    22%     35%                                                    ______________________________________                                    

We claim:
 1. A compound of the formula: ##STR5## wherein: A representshydroxyl, O-alkyl or O-benzyl, B represents --CH₂ -- or --CH₂ CH₂ -- andb is 0 or 1, and their pharmaceutically acceptable acid addition salts.2. The compound of claim 1, wherein A represents a 4-positionsubstituent.
 3. The compound of claim 1, wherein A represents hydroxyl.4. The compound of claim 2, wherein A represents hydroxyl.
 5. A compoundselected from the group consisting ofN-(4-hydroxyphenylacetyl)-spermine, and N-(4-hydroxybenzoyl)-spermineand pharmaceutically acceptable acid addition salts thereof.
 6. A methodof treating a patient suffering from cerebral ischaemia, which comprisesadministering to such a patient a therapeutically effective amount of acompound claimed in claim
 1. 7. The method of claim 6, wherein Arepresents a 4-position substituent.
 8. The method of claim 6, wherein Arepresents hydroxyl.
 9. The method of claim 7, wherein A representshydroxyl.
 10. The method of treating a patient suffering from cerebralischaemia, which comprises administering to such a patient atherapeutically effective amount of a compound claimed in claim
 5. 11. Apharmaceutical composition for the treatment of cerebral ischaemia,comprising a therapeutically effective amount of a compound claimed inclaim 1 together with a pharmaceutically acceptable diluent or carrier.12. The composition of claim 1, wherein A represents hydroxyl in the4-position.
 13. A pharmaceutical composition for the treatment ofcerebral ischaemia, comprising a therapeutically effective amount of acompound claimed in claim 5 together with a pharmaceutically acceptablediluent or carrier.
 14. The method of claim 6, wherein the patient issuffering from a stroke.
 15. The method of claim 10, wherein the patientis suffering from a stroke.